This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Fab-7 boundary functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax Complex from early embryogenesis through to the adult stage. Although Fab-7 is only required for the proper development of a single posterior parasegment, it is active in all tissues and stages of development that have been examined. In this respect Fab-7 resembles conventional constitutive boundaries in flies and other eukaryotes that act through ubiquitous cis-elements and trans-acting factors. The Fab-7 boundary spans a DNA sequence of ~1.2 kb and contains 3 chromatin specific nuclease hypersensitive sites. We have examined the functioning of cis-acting sequences in the largest of these hypersensitive sites, HS1, in detail. Though Fab-7 boundary activity is constitutive, we found that HS1 is divided into sub-elements whose boundary activity is restricted to only a specific stage in development. One sub-element, which is located on the proximal side of HS1, is active only in the early embryo. A second sub-element, which is located in the middle of HS1, is not active in early embryos;however, it becomes active mid-way through embryogenesis and then remains active through to the adult stage. A third sub-element on the distal side of HS1 confers boundary activity from early through mid-embryogenesis but not later. Through this project, we identified candidate proteins that are likely to confer this early-stage specific boundary activity, and then found that these proteins make a heterogeneous protein complex to bind the target sequence. We also would like to identify the trans-acting factors that confer other stage specific boundary activity in each.